Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism in Photorhabdus luminescens

The enteric gamma‐proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post‐exponential (stationary) phase. During post‐exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well‐described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.     

Insect pathogenicity islands in the insect pathogenic bacterium Photorhabdus

Abstract. Genomic islands are regions of the bacterial genome responsible for unique aspects of bacterial behaviour, such as host symbiosis and pathogenicity. Where such regions are involved in pathogenesis, they are termed pathogenicity islands (PAIs). Photorhabdus luminescens is an insect pathogen that spends part of its life in symbiosis with a nematode and part of its life as an insect pathogen. Here, several novel PAIs from P. luminescens ssp. akhurstii strain W14 are described that encode factors involved apparently in both nematode symbiosis and insect pathogenicity. The structures of these islands are compared with those found in mammalian pathogens, and the potential cross‐talk between virulence factors used against invertebrates and those used against vertebrates is discussed.     

Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens

Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.     

For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

TCA cycle activity in Staphylococcus aureus is essential for iron‐regulated synthesis of staphyloferrin A,but not staphyloferrin B: the benefit of a second citrate synthase

Staphylococcus aureus elaborates two citrate‐containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron‐restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron‐regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron‐restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB‐deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron‐restricted media, and is significantly more impaired for virulence than either of the single‐deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron‐restricted growth, without need of TCA cycle activity.     

Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture

Photorhabdus luminescens are bacterial symbionts of entomopathogenic nematodes of the genus Heterorhabditis. The bacto-helminthic complexes are used in biocontrol of insect pests in cryptic environments. For in vitro production, liquid media are incubated with P. luminescens for 24 h prior to the inoculation of nematode dauer juveniles. The nematodes develop to self-fertilizing hermaphrodites and produce offspring. The exit from the developmentally arrested dauer stage (recovery) is a response to a yet undescribed food signal. Major process instability is caused by low and unsynchronized recovery of the dauers. In living insects, dauer recovery is approximately 95% within 1 day. In liquid cultures of P. luminescens the recovery is spread over several days and varies between 0 and 81%. In complex culture media no food signal was detected. A food signal is produced by P. luminescens and excreted into the culture medium. The maximum food signal production was recorded during the late exponential growth phase. Compared to the food signal found in insects, the efficacy of the bacterial signal is much lower. The reasons for the variable activity of the bacterial food signal and its function during the nematode life cycle are discussed. Received: 13 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

The global regulator Crc modulates metabolism,susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross‐talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high‐throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing‐regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta‐lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.     

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering

Mannheimia succiniciproducens, a capnophilic gram‐negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome‐scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering (‘EMC’ analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid‐overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH‐dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co‐expressing mdh alone and both zwf and mdh genes are subjected to fed‐batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.     

Genomic islands in Photorhabdus

Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.     

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg²⁺ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l- malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.     

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

Virulence and competitive ability in an obligately killing parasite

Mixed infections are thought to have a major influence on the evolution of parasite virulence. During a mixed infection, higher within‐host parasite growth is favored under the assumption that it is critical to the competitive success of the parasite. As within‐host parasite growth may also increase damage to the host, a positive correlation is predicted between virulence and competitive success. However, when parasites must kill their hosts in order be transmitted, parasites may spend energy on directly attacking their host, even at the cost of their within‐host growth. In such systems, a negative correlation between virulence and competitive success may arise. We examined virulence and competitive ability in three sympatric species of obligately killing nematode parasites in the genus Steinernema. These nematodes exist in a mutualistic symbiosis with bacteria in the genus Xenorhabdus. Together the nematodes and their bacteria kill the insect host soon after infection, with reproduction of both species occurring mainly after host death. We found significant differences among the three nematode species in the speed of host killing. The nematode species with the lowest and highest levels of virulence were associated with the same species of Xenorhabdus, indicating that nematode traits, rather than the bacterial symbionts, may be responsible for the differences in virulence. In mixed infections, host mortality rate closely matched that associated with the more virulent species, and the more virulent species was found to be exclusively transmitted from the majority of coinfected hosts. Thus, despite the requirement of rapid host death, virulence appears to be positively correlated with competitive success in this system. These findings support a mechanistic link between parasite growth and both anti‐competitor and anti‐host factors.     

Stability of entomopathogenic bacteria, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>, during in vitro culture

The entomopathogenic nematode–bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus luminescens Cell Populations

Phenotypic heterogeneity in bacterial cell populations allows genetically identical organisms to different behavior under similar environmental conditions. The Gram‐negative bacterium Photorhabdus luminescens is an excellent organism to study phenotypic heterogeneity since their life cycle involves a symbiotic interaction with soil nematodes as well as a pathogenic association with insect larvae. Phenotypic heterogeneity is highly distinct in P. luminescens. The bacteria exist in two phenotypic forms that differ in various morphologic and phenotypic traits and are therefore distinguished as primary (1°) and secondary (2°) cells. The 1 cells are bioluminescent, pigmented, produce several secondary metabolites and exo-enzymes, and support nematode growth and development. The 2° cells lack all these 1°-specific phenotypes. The entomopathogenic nematodes carry 1° cells in their upper gut and release them into an insect's body after slipping inside. During insect infection, up to the half number of 1° cells undergo phenotypic switching and convert to 2° cells. Since the 2° cells are not able to live in nematode symbiosis any more, they cannot re-associate with their symbiosis partners after the infection and remain in the soil. Phenotypic switching in P. luminescens has to be tightly regulated since a high switching frequency would lead to a complete break-down of the nematode-bacteria life cycle. Here, we present the main regulatory mechanisms known to-date that are important for phenotypic switching in P. luminescens cell populations and discuss the biological reason as well as the fate of the 2° cells in the soil.